AI-based structure prediction empowers integrative structural analysis of human nuclear pores

Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120-megadalton architecture remains incompletely understood. Here, we report a 70-megadalton model of the humanNPC scaffold with explicit membrane and in multiple conformational states. We combined artificial intelligence (AI)–based structure prediction with in situ and in cellulo cryo–electron tomography and integrative modeling. We show that linker nucleoporins spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulationssuggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated within situ structural biology to understand subcellular architecture across spatial organization levels

3D super-resolution fluorescence microscopy maps the variable molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of over 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of 3D super-resolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of over ten thousand individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ

Pore timing:the evolutionary origins of the nucleus and nuclear pore complex

The name “eukaryote” is derived from Greek, meaning “true kernel”, and describes the domain of organisms whose cells have a nucleus. The nucleus is thus the defining feature of eukaryotes and distinguishes them from prokaryotes (Archaea and Bacteria), whose cells lack nuclei. Despite this, we discuss the intriguing possibility that organisms on the path from the first eukaryotic common ancestor to the last common ancestor of all eukaryotes did not possess a nucleus at all—at least not in a form we would recognize today—and that the nucleus in fact arrived relatively late in the evolution of eukaryotes. The clues to this alternative evolutionary path lie, most of all, in recent discoveries concerning the structure of the nuclear pore complex. We discuss the evidence for such a possibility and how this impacts our views of eukaryote origins and how eukaryotes have diversified subsequent to their last common ancestor

Structural studies on human RZZ and Cln3p

Die vorliegende Dissertation ist in zwei Thematiken unterteilt: 1.Molekulare Mechanismen der Segregation von Chromosomen: Die Segregation der Chromosomen ist ein zentraler Prozess während der Zellteilung. Dieser wird durch hierarchische Proteinkomplexe reguliert, die Kinetochore. Sie kontrollieren die synchrone Teilung der Schwesterchromatiden in höheren Eukaryoten in einem Prozess, der als spindle assembly checkpoint (SAC) bezeichnet wird. Der 800 kDa große ROD-Zw10-Zwilch (RZZ) Proteinkomplex besitzt eine Schlüsselfunktion in der Formierung des SAC sowie der Anheftung der Mikrotubuli an die Kinetochore. Diese Arbeit beschreibt die erste mittels Kryo-Elektronenmikroskopie bestimmte hochaufgelöste Struktur des RZZ Komplexes. Die Organisation der Bestandteile des Komplexes wurde durch Einbeziehung diverser biochemischer Methoden charakterisiert. Diese Arbeit stellt den ersten Schritt für ein strukturelles Verständnis des SAC dar und ermöglicht hierdurch ein besseres Verständnis der komplexen Signalwege, welche die Zellteilung steuern. 2. Molekulare Grundlagen der Batten-Krankheit: Das Ceroid-lipofuscinosis neuronal-3 (cln3) Gen kodiert für ein Transmembranprotein (Cln3p). Mutationen dieses Proteins bilden die Grundlage für die Batten-Krankheit, die am weitesten verbreitete aller neuronalen Ceroid-Lipofuscinosen (NCL). Diese vererbbaren neuronalen Krankheiten sind durch den Verlust der Sehkraft, myoklonische Anfälle, Verlust der geistigen Fähigkeiten sowie Störungen der motorischen Fertigkeiten charakterisiert. In dieser Arbeit wird die erste erfolgreiche Expression und Reinigung des humanen Cln3p mittels eines Baculovirus-Insektenzellen-Expressionssystems beschrieben. Initiale biochemische Charakterisierungen der posttranslationalen Modifikationen des Proteins wurden durchgeführt. Weiterhin wurden erste Versuche zum Aufklärung der Proteinfunktion und der Struktur des Proteins begonnen.The work presented in this thesis can be divided into two topics: 1. Molecular mechanism of chromosome segregation: Chromosome segregation is a central process in cell division. Hierarchical protein assemblies called kinetochores navigate this process. They monitor the synchronous separation of sister chromatids in higher eukaryotes by a pathway commonly referred to as spindle assembly checkpoint (SAC). The 800 kDa ROD-Zw10-Zwilch (RZZ) complex is a key player both in SAC and formation of kinetochore – microtubule attachments. This work highlights the first high-resolution structure of the RZZ complex determined by cryo-electron microscopy. The organization of constituent proteins in the complex has been understood by interweaving various biochemical methods. This study is essentially the first step in understanding the SAC from a structural perspective and enables to use hints from the structure in understanding the complex signaling process that drives cell division. 2. Molecular basis of the Batten disease: Ceroid – lipofuscinosis neuronal-3 (cln3) gene encodes a trans-membrane protein (Cln3p) mutations in which cause Batten’s disease. Batten disease is the most common of all neuronal ceroid-lipofuscinoses (NCL, genetically inherited neuronal disorders) characterized by loss of vision, myoclonic seizures, loss of cognitive function, motor dysfunction. This work reports the first expression and purification of human Cln3p from the baculovirus driven insect cell expression system. Initial biochemical characterization of the protein is also reported. Efforts to understand the protein function have been initialized and variety of techniques used to understand the structure of the protein

From the resolution revolution to evolution: structural insights into the evolutionary relationships between vesicle coats and the nuclear pore

Nuclear pores and coated vesicles are elaborate multi-component protein complexes that oligomerize on membranes, and stabilize or induce membrane curvature. Their components, nucleoporins and coat proteins, respectively, share similar structural folds and some principles of how they interact with membranes. The protocoatomer hypothesis postulates that this is due to divergent evolution from a common ancestor. It therefore has been suggested that nucleoporins and coat proteins have similar higher order architectures. Here, we review recent work that relied on technical advances in cryo-electron microscopy and integrative structural biology to take a fresh look on how these proteins form membrane coats in situ. We discuss the relationship between the architectures of nuclear pores and coated vesicles, and their evolutionary origins

Selective autophagy degrades nuclear pore complexes

Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3-7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope

Proteasomes tether to two distinct sites at the nuclear pore complex

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC